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Title: Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Lastly, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalentmore » “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.« less
Authors:
 [1] ;  [2] ;  [2] ;  [2] ;  [2] ;  [2]
  1. Beckman Research Institute of City of Hope, Duarte, CA (United States); Thomas Jefferson Univ., Philadelphia, PA (United States)
  2. Beckman Research Institute of City of Hope, Duarte, CA (United States)
Publication Date:
OSTI Identifier:
1135749
Report Number(s):
SLAC-REPRINT--2014-168
Journal ID: ISSN 0027-8424
Grant/Contract Number:
AC02-76SF00515
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 110; Journal Issue: 43; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES protein engineering; molecular recognition; cancer